Nuclear import receptors as modifiers of TDP-43 phase transition and toxicity in FTD/ALS - The goal of this project is to gain a detailed mechanistic understanding of how nuclear-import receptors (NIRs) can prevent and reverse the cytoplasmic mislocalization and accumulation of insoluble protein aggregates of the RNA-binding protein TDP-43 in the pathogenesis of common neurodegenerative disorders. TDP-43 pathology is a hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), but is also commonly found in Alzheimer’s disease (AD) and other AD-related dementias (ADRDs), marking it as a high priority target for therapy development. Our labs have discovered that 1) TDP-43 pathology is characterized by the co-aggregation of TDP-43 with FG nucleoporins (FG-Nups) in the cytoplasm, causing nucleocytoplasmic transport defects and 2) NIRs can reduce the aberrant phase transition of TDP-43 and other disease-causing RNA-binding proteins with prion-like domains. Our data demonstrate that specific NIRs can reverse the hallmarks of TDP-43 proteinopathy in cellular and animal models of FTD and ALS. Based on our findings, we propose a novel non-canonical role for NIRs as potent molecular chaperones that are recruited by FG-Nups into pathological TDP-43 aggregates, where they act to to reverse the aberrant phase transition and restore the normal nuclear localization and splicing functions of TDP-43, suggesting a promising new strategy for therapeutic intervention. To test this hypothesis, our team of experts in cellular and animal models of FTD (an ADRD) and ALS, as well as structural biology approaches, will use cutting edge in vitro and in vivo methods to gain a detailed mechanistic understanding of how NIRs restore solubility, nuclear localization and normal function of TDP-43; how NIRs reduce neurodegeneration; how NIR dysfunction contributes to human disease; and how we can use this knowledge of NIR functions to develop future therapeutic strategies for TDP-43 proteinopathies. Our specific aims are: (i) to determine how NIRs restore proper TDP-43 localization and reduce aberrant phase transition in vitro, using a combination of advanced neuronal cell culture models of FTD and ALS and biochemical characterization of TDP-43 liquid-liquid phase separation (LLPS) and fibrillization, and rational protein engineering approaches to potentiate the chaperone activity of NIRs; and (ii) to identify how NIRs reduce TDP-43-dependent neurodegeneration in cellular and animal models in vivo, employing Drosophila, organotypic slice cultures and somatic brain transgenesis in TDP-43 proteinopathy mouse models, and clarifying the nature of NIR pathology in ALS and FTD (an ADRD) patient brain tissue. Successful outcome of this project will clarify the role of NIRs in regulating TDP-43 phase transition during pathogenesis and how this activity can restore normal localization and cellular function of TDP-43. This knowledge will be critical for developing new therapeutic strategies to target aberrant phase separation in ALS, FTD, and other devastating AD-related neurodegenerative disorders with TDP-43 proteinopathy.
